Light-harvesting Complex II Proteins Research Articles

Functions and Evolution of Lhcb Isoforms Composing LHCII, the Major Light Harvesting Complex of Photosystem II of Green Eukaryotic Organisms.

Oxygenic photosynthesis provides energy and oxygen for almost all forms of life on earth. This process is based on the energy of photons, which is used to split water and use its electrons to reduce carbon atoms to create organic molecules and thus fix the light energy into a chemical form. Two photosytems working in series are involved in light harvesting and conversion. Both are multi-protein supercomplexes composed of a core complex, where the photochemical reaction takes place, and an antenna system involved in light harvesting. In plants and green algae, the antenna of photosystem II, the photosynthetic complex involved in water splitting, comprises the Light Harvesting Complex II (LHCII) trimers, the most abundant membrane protein on earth. LHCII is composed of highly conserved Lhcb isoforms and all green organisms count a high number of Lhcb. In vascular plants they are classified in three distinct subclasses, Lhcb1, 2 and 3, while in algae and non-vascular plants, these isoforms are less differentiated and called Lhcbm proteins. In this review, we compare LHCII proteins of different organisms, from green algae to angiosperms, and discuss the role of the modifications that occurred through evolution. We highlight the various functions of the different isoforms in photosynthesis, ranging from light harvesting, a common role to all these proteins, to regulations of photosynthesis that rely on specific isoforms.

Grana-Localized Proteins, RIQ1 and RIQ2, Affect the Organization of Light-Harvesting Complex II and Grana Stacking in Arabidopsis.

Grana are stacked thylakoid membrane structures in land plants that contain PSII and light-harvesting complex II proteins (LHCIIs). We isolated two Arabidopsis thaliana mutants, reduced induction of non-photochemical quenching1 (riq1) and riq2, in which stacking of grana was enhanced. The curvature thylakoid 1a (curt1a) mutant was previously shown to lack grana structure. In riq1 curt1a, the grana were enlarged with more stacking, and in riq2 curt1a, the thylakoids were abnormally stacked and aggregated. Despite having different phenotypes in thylakoid structure, riq1, riq2, and curt1a showed a similar defect in the level of nonphotochemical quenching of chlorophyll fluorescence (NPQ). In riq curt1a double mutants, NPQ induction was more severely affected than in either single mutant. In riq mutants, state transitions were inhibited and the PSII antennae were smaller than in wild-type plants. The riq defects did not affect NPQ induction in the chlorophyll b-less mutant. RIQ1 and RIQ2 are paralogous and encode uncharacterized grana thylakoid proteins, but despite the high level of identity of the sequence, the functions of RIQ1 and RIQ2 were not redundant. RIQ1 is required for RIQ2 accumulation, and the wild-type level of RIQ2 did not complement the NPQ and thylakoid phenotypes in riq1 We propose that RIQ proteins link the grana structure and organization of LHCIIs.

Characterization of light-dependent regulation of state transitions in gymnosperms.

The goal of this study was to characterize the light-dependent regulation of state transitions in gymnosperms. Two species of conifer were examined: eastern white pine (Pinus strobus L.) and white spruce [Picea glauca (Moench) Voss], as well as the angiosperm pumpkin (Cucurbita pepo L. subsp. pepo). Both diurnal time courses in the field and manipulated light experiments in growth chambers were conducted. Results from chlorophyll fluorescence analysis indicated that pumpkin was able to use a larger fraction of absorbed light to drive photochemistry and retain a lower reduction state at a given light intensity relative to the conifers. Results from western blots using anti-phosphothreonine demonstrate that in field conditions, conifers maintained higher light-harvesting complex II (LHCII) phosphorylation than pumpkin; however, this was likely due to a more variable light environment. Manipulated light experiments showed that general patterns of light-dependent LHCII phosphorylation were similar in conifers and pumpkin, with low levels of LHCII phosphorylation occurring in darkness and maximal levels occurring in low light conditions. However, high light-dependent dephosphorylation of LHCIII appears to be regulated differently in conifers, with conifers maintaining phosphorylation of LHCII proteins at higher excitation pressure compared with pumpkin. Additionally, spruce needles maintained relatively high phosphorylation of LHCII even in very high light conditions. Our results suggest that this difference in dephosphorylation of LHCII may be due to differences in the stromal redox status in spruce relative to pine and pumpkin.

Synthesis and Functional Reconstitution of Light-Harvesting Complex II into Polymeric Membrane Architectures.

One of most important processes in nature is the harvesting and dissipation of solar energy with the help of light-harvesting complex II (LHCII). This protein, along with its associated pigments, is the main solar-energy collector in higher plants. We aimed to generate stable, highly controllable, and sustainable polymer-based membrane systems containing LHCII-pigment complexes ready for light harvesting. LHCII was produced by cell-free protein synthesis based on wheat-germ extract, and the successful integration of LHCII and its pigments into different membrane architectures was monitored. The unidirectionality of LHCII insertion was investigated by protease digestion assays. Fluorescence measurements indicated chlorophyll integration in the presence of LHCII in spherical as well as planar bilayer architectures. Surface plasmon enhanced fluorescence spectroscopy (SPFS) was used to reveal energy transfer from chlorophyll b to chlorophyll a, which indicates native folding of the LHCII proteins.

Plants Actively Avoid State Transitions upon Changes in Light Intensity: Role of Light-Harvesting Complex II Protein Dephosphorylation in High Light

Photosystem II (PSII) core and light-harvesting complex II (LHCII) proteins in plant chloroplasts undergo reversible phosphorylation upon changes in light intensity (being under control of redox-regulated STN7 and STN8 kinases and TAP38/PPH1 and PSII core phosphatases). Shift of plants from growth light to high light results in an increase of PSII core phosphorylation, whereas LHCII phosphorylation concomitantly decreases. Exactly the opposite takes place when plants are shifted to lower light intensity. Despite distinct changes occurring in thylakoid protein phosphorylation upon light intensity changes, the excitation balance between PSII and photosystem I remains unchanged. This differs drastically from the canonical-state transition model induced by artificial states 1 and 2 lights that concomitantly either dephosphorylate or phosphorylate, respectively, both the PSII core and LHCII phosphoproteins. Analysis of the kinase and phosphatase mutants revealed that TAP38/PPH1 phosphatase is crucial in preventing state transition upon increase in light intensity. Indeed, tap38/pph1 mutant revealed strong concomitant phosphorylation of both the PSII core and LHCII proteins upon transfer to high light, thus resembling the wild type under state 2 light. Coordinated function of thylakoid protein kinases and phosphatases is shown to secure balanced excitation energy for both photosystems by preventing state transitions upon changes in light intensity. Moreover, proton gradient regulation5 (PGR5) is required for proper regulation of thylakoid protein kinases and phosphatases, and the pgr5 mutant mimics phenotypes of tap38/pph1. This shows that there is a close cooperation between the redox- and proton gradient-dependent regulatory mechanisms for proper function of the photosynthetic machinery.

Revisiting the Supramolecular Organization of Photosystem II in Chlamydomonas reinhardtii

Photosystem II (PSII) is a multiprotein complex that splits water and initiates electron transfer in photosynthesis. The central part of PSII, the PSII core, is surrounded by light-harvesting complex II proteins (LHCIIs). In higher plants, two or three LHCII trimers are seen on each side of the PSII core whereas only one is seen in the corresponding positions in Chlamydomonas reinhardtii, probably due to the absence of CP24, a minor monomeric LHCII. Here, we re-examined the supramolecular organization of the C. reinhardtii PSII-LHCII supercomplex by determining the effect of different solubilizing detergents. When we solubilized the thylakoid membranes with n-dodecyl-β-D-maltoside (β-DM) or n-dodecyl-α-D-maltoside (α-DM) and subjected them to gel filtration, we observed a clear difference in molecular mass. The α-DM-solubilized PSII-LHCII supercomplex bound twice more LHCII than the β-DM-solubilized supercomplex and retained higher oxygen-evolving activity. Single-particle image analysis from electron micrographs of the α-DM-solubilized and negatively stained supercomplex revealed that the PSII-LHCII supercomplex had a novel supramolecular organization, with three LHCII trimers attached to each side of the core.

CP29, a Monomeric Light-harvesting Complex II Protein, Is Essential for State Transitions in Chlamydomonas reinhardtii

In oxygen-evolving photosynthesis, the two photosystems, photosystem I (PSI) and photosystem II (PSII), function in parallel, and their excitation levels must be balanced to maintain an optimal photosynthetic rate under various light conditions. State transitions balance excitation energy between the two photosystems by redistributing light-harvesting complex II (LHCII) proteins. Here we describe two RNA interference (RNAi) mutants of the green alga Chlamydomonas reinhardtii with one of the minor monomeric LHCII proteins, CP29 or CP26, knocked down. These two proteins have been identified in PSI-LHCI supercomplexes that harbor mobile LHCII proteins from PSII under a state where PSII is preferentially excited (State 2). We show that both the CP29 and CP26 RNAi mutants undergo reductions in the PSII antenna size during a transition from State 1 (a state where PSI is preferentially excited) to State 2, as reflected by nonphotochemical quenching of fluorescence, low temperature fluorescence spectra, and functional absorption cross-section. However, the undocked LHCIIs from PSII do not re-associate with PSI in the CP29-RNAi (b4i) mutant because the antenna size of PSI was not complementary increased. The mobile LHCIIs in the CP26-RNAi (b5i) mutant, however, re-associate with PSI, whose PSI-LHCI/II supercomplex is visualized on a sucrose density gradient. This study clarifies that CP29, not CP26, is an essential component in state transitions and demonstrates that CP29 is crucial when mobile LHCIIs re-associate with PSI under State 2 conditions.

Molecular Remodeling of Photosystem II during State Transitions inChlamydomonas reinhardtii

State transitions, or the redistribution of light-harvesting complex II (LHCII) proteins between photosystem I (PSI) and photosystem II (PSII), balance the light-harvesting capacity of the two photosystems to optimize the efficiency of photosynthesis. Studies on the migration of LHCII proteins have focused primarily on their reassociation with PSI, but the molecular details on their dissociation from PSII have not been clear. Here, we compare the polypeptide composition, supramolecular organization, and phosphorylation of PSII complexes under PSI- and PSII-favoring conditions (State 1 and State 2, respectively). Three PSII fractions, a PSII core complex, a PSII supercomplex, and a multimer of PSII supercomplex or PSII megacomplex, were obtained from a transformant of the green alga Chlamydomonas reinhardtii carrying a His-tagged CP47. Gel filtration and single particles on electron micrographs showed that the megacomplex was predominant in State 1, whereas the core complex was predominant in State 2, indicating that LHCIIs are dissociated from PSII upon state transition. Moreover, in State 2, strongly phosphorylated LHCII type I was found in the supercomplex but not in the megacomplex. Phosphorylated minor LHCIIs (CP26 and CP29) were found only in the unbound form. The PSII subunits were most phosphorylated in the core complex. Based on these observations, we propose a model for PSII remodeling during state transitions, which involves division of the megacomplex into supercomplexes, triggered by phosphorylation of LHCII type I, followed by LHCII undocking from the supercomplex, triggered by phosphorylation of minor LHCIIs and PSII core subunits.

Phosphorylation-dependent regulation of excitation energy distribution between the two photosystems in higher plants

Phosphorylation-dependent movement of the light-harvesting complex II (LHCII) between photosystem II (PSII) and photosystem I (PSI) takes place in order to balance the function of the two photosystems. Traditionally, the phosphorylatable fraction of LHCII has been considered as the functional unit of this dynamic regulation. Here, a mechanical fractionation of the thylakoid membrane of Spinacia oleracea was performed from leaves both in the phosphorylated state (low light, LL) and in the dephosphorylated state (dark, D) in order to compare the phosphorylation-dependent protein movements with the excitation changes occurring in the two photosystems upon LHCII phosphorylation. Despite the fact that several LHCII proteins migrate to stroma lamellae when LHCII is phosphorylated, no increase occurs in the 77 K fluorescence emitted from PSI in this membrane fraction. On the contrary, such an increase in fluorescence occurs in the grana margin fraction, and the functionally important mobile unit is the PSI–LHCI complex. A new model for LHCII phosphorylation driven regulation of relative PSII/PSI excitation thus emphasises an increase in PSI absorption cross-section occurring in grana margins upon LHCII phosphorylation and resulting from the movement of PSI–LHCI complexes from stroma lamellae and subsequent co-operation with the P-LHCII antenna from the grana. The grana margins probably give a flexibility for regulation of linear and cyclic electron flow in plant chloroplasts.

Identification of the mobile light-harvesting complex II polypeptides for state transitions in Chlamydomonas reinhardtii.

State transition in photosynthesis is a short-term balancing mechanism of energy distribution between photosystem I (PSI) and photosystem II (PSII). When PSII is preferentially excited (state 2), a pool of mobile light-harvesting complex II (LHCII) antenna proteins is thought to migrate from PSII to PSI, but biochemical evidence for a physical association between LHCII proteins and PSI in state 2 is weak. Here, using the green alga Chlamydomonas reinhardtii, which has a high capacity for state transitions, we report the isolation of PSI-light-harvesting complex I (LHCI) super-complexes from cells locked into state 1 and state 2. We solubilized the thylakoid membranes with a mild detergent, separated the proteins by sucrose density gradient centrifugation, and subjected gradient fractions to gel-filtration chromatography. Three LHCII polypeptides were associated with a PSI-LHCI supercomplex only in state 2; we identified them as two minor monomeric LHCII proteins (CP26 and CP29) and one previously unreported major LHCII protein type II, or LhcbM5. These three LHCII proteins, in addition to the major trimeric LHCII proteins, were phosphorylated upon transition to state 2. The corresponding phylogenetic tree indicates that among the LHCII proteins associated with PSII, these three LHCII proteins are the most similar to the LHC proteins for PSI (LHCI). Our results are important because CP26, CP29, and LhcbM5, which have been viewed as belonging solely to the PSII complex, are now postulated to shuttle between PSI and PSII during state transitions, thereby acting as docking sites for the trimeric LHCII proteins in both PSI and PSII.

The Flow of Excitation Energy in LHCII Monomers: Implications for the Structural Model of the Major Plant Antenna

Spectral and kinetic information on energy transfer within the light-harvesting complex II (LHCII) monomer was obtained from this subpicosecond transient absorption study, by using selective excitation (663, 669, 672, 678, and 682 nm) of various Chl a absorption bands and detecting the induced changes over the entire Q y region (650–700 nm). It is shown that transfer from the pigment(s) absorbing around 663 nm to the low energy ones occurs in 5 ± 1 ps, whereas the 670-nm excitation is delivered to the same “destination” in two phases (0.30 ± 0.05 ps, and 12 ± 2 ps), and a fast equilibration (lifetime 0.45 ± 0.05 ps) takes place within the main absorption band (675–680 nm). From comparison with results from similar time-resolved measurements on trimeric samples, it can be concluded that the intramonomeric energy transfer completely determines the spectral equilibration observed in native LHCII complexes. To correlate the measured lifetimes and their associated spectra with the pigment organization within the available structural model of LHCII (Kühlbrandt et al. 1994. Nature. 367:614–621), extensive but straightforward theoretical modeling was used. Thus it is demonstrated that the pigment assignment (Chl a or Chl b) given by Kühlbrandt and co-workers cannot simultaneously describe the dichroic spectra and the transient absorption results for the rather homologous LHCII and CP29 proteins. A more recent assignment for CP29, in which a Chl b molecule (“Chl b5”) is identified as a Chl a (Dr. R. Bassi, personal communication), leads to a much better description of both CP29 and LHCII. Furthermore, the orientations of the transition dipole moments, which have not been obtained in the crystal structure, are now assigned for most of the Chl’s.

Chloroplast Response in Dunaliella salina to Irradiance Stress (Effect on Thylakoid Membrane Protein Assembly and Function).

The chloroplast response in the green alga Dunaliella salina to irradiance stress was investigated. Cells were grown under low light (LL) at 100 [mu]mol photons m-2 s-1 or high light (HL) at 2000 [mu]mol photons m-2 s-1 incident intensity. LL-grown cells had a low chlorophyll (Chl) a/b ratio, an abundance of light-harvesting complex II proteins (LHC-II), and a large Chl antenna size. HL-grown cells had a higher Chl a/b ratio, relatively fewer LHC-II, and a small Chl antenna size. The more abundant higher molecular mass subunits of the LHC-II (approximately 31 kD) were selectively depleted from the thylakoid membrane of HL-grown cells. Light-shift experiments defined the kinetics of change in the subunit composition of the LHC-II and suggested distinct mechanisms in the acclimation of thylakoids to HL or LL conditions. The results showed that irradiance exerts a differential regulation on the expression of various Lhcb genes. The specific polyclonal antibodies used in this work, raised against the purified LHC-II, cross-reacted with a polypeptide of approximately 20 kD in HL-grown samples. In this work we examined the dynamics of induction of this novel protein and discuss its function in terms of a chloroplast response to the level of irradiance.